Removal of substances from a culture by the employment of electro-endosmosis



United States Patent REMOVAL OF SUBSTANCES FROM A CULTURE EMPLOYMENT OF ELECTRO-ENDOS- Edwin C. Griesbach, La Grange, iii.

No Drawing. Application February 6, 1956,

. Serial No. 563,809

8 Claims. (Cl. 195-77) This application is a continuation in part of my copending applications Serial No. 729,665, filed February 19, 1947, for Method of Low Alcohol Yeast Manufacture, now abandoned, and Serial No. 253,455, filed October 26, 1951, now abandoned, for Method of Employing Ionic Migration'in a Culture of Micro-Organisms, and Serial No. 438,329, filed June 21, 1954, now abandoned, for Removal of Substances From a Culture by the Employment of Electro-Endosmosis.

This invention relates to a method of controlling a biological process by removing a constituent of an innculated-nutrient solution during the culture of the inoculum.

The present invention is useful to many industrial processes requiring the culture of large quantities of micro-organisms; as for example, yeasts, molds and bacteria for the following purposes: products of fermentation, production of breadstufi, pharmaceutical ingredients and warfare.

The chief object of this invention is to provide a method of selectively removing a component and/ or product from an inoculated-nutrient solution in an efficient, convenient and economical manner by the employment of electroendosmosis, particularly during the culture of the inoculum. In some cases the actual yield may be increased in this manner, for instance when the action of the enzyme or bacillus producing the fermentation is deleteriously affected by a high concentration of the product, as in lactic fermentation.

As a further object, by the hereinafter more fully described method of removing a component of an inoculated-nutrient solution there may be obtained a substantial increase in the reproduction of the inoculum relative to a given amount of nutrient material.

The following will describe specific means for the use of my invention; however, certain objects will be apparent from a full consideration of the specification and claims by those familar with biological processes. Also, while I shall describe specific means by which my method can be carried out, it is to be understood that the invention is not to be limited to any specific organisms, particular type of apparatus, exact electrolyte and/ or nutrient formulas, exact current densities, nor definite methods of procedure, as these can all be varied within wide limits without departing from the scope of the present invention.

I use an electrolytic cell which is divided into two parts by a finely-porous diaphragm which must be sufliciently porous to permit the migration of ions, and at the same time sufliciently dense to prevent the diffusion of the electrolyte into the other section of the cell. One section of this cell (the cathodic section) contains a suitable cathode and a means for agitating the catholyte which also serves as an inoculated-nutrient solution and may hereinafter be termed the inoculated-nutrient solution or culture; the other section of the cell (the anode section)-containsan anode of suitable material (such Patented Aug. 13, 1957 as a carbon rod and in this anodic compartment is placed an aqueous solution which contains a suitable electrolyte and this electrolytic solution will hereinafter be termed the anolyte. I

In most cases an increased benefit is obtained by periodically effecting electro-endosmosis so adjusted that the time lengths of the periods are sufiicient to remove a component and/ or product from the culture. According to the herein examples, relatively small successive portions of the culture are intermittently subjected to electro-endosmosis, as the process progresses, in a manner to dialytically separate a component and/ or product from the culture which serves as a catholyte, by effecting the migration of anions into the confined anolyte, the depth or pressure of which can be adjusted, and therein retain said component and/or product.

One of the advantages of electro-endosmosis, as taught by the contemplated invention, is the provision of a means for the maintenance of a more optimum environment for the inoculum for the reason that, as the process progresses, the presence in the inoculated-nutrient solution of a deleterious degree of acid resulting from cumulative substances of an eliminated and/ or nonassimilable char acter can be prevented by the removal of this toxic acid with the employment of ionic migration, thus making the remaining culture medium more assimilable for the inoculum. Also, similar benefits are obtainable for the culture of microorganisms wherein mineral salts, such as ammonium sulphate, are present and the organisms selectively utilize basic components thereof for cell growth and by so doing there remains a deleterious degree of mineral acid in the nutrient solution which may be dialytically removed in the form of anions by their migration into the anodic cell.

In some cases an increase in the cell count and/or productivity of the organisms canbe obtained by alternating periods of electro-endosmosis with stationary periods so adjusted that the time lengths of the periods of electro-endosmosis approximately coincide with the time lengths of generative cessation of the organisms. That is to say, electro-endosmosis is periodically effected during the periods of generative inactivity or rest periods between regeneration of the microorganisms.

In the practice of this invention I have found it advantageous to revolve the inoculated-nutrient solution in a vessel which is tall as compared to its width, so as to transiently circulate relatively small portions of the culture through a localized current of electricity being introduced with the use of a finely porous anodic diaphragm which is disposed in the bottom half of the vessel. This circulation is to be of sufficient velocity to counteract any tendency of the organisms to adhere to a diaphragm by the washing action of the moving inoculated-nutrient fluid, thereby maintaining the organisms in suspension throughout the nutrient solution. In addition, the suspension of the organisms throughout the nutrient solution may be facilitated by the use of a current of electricity which has a ripple or is intermittent. It will be perceived that, by a rapid cyclical circulation the organisms are subjected to a current of electricity and the heat thereby produced for only very short periods of time, which precludes any harmful effect of the electricity on the organisms.

Example I For the employment of the present invention wherein yeast is propagated, a dilute solution was prepared which contained a carbohydrate in the form of refined cane sugar with an essential amount of organic nitrogen in the form of malt sprout extract and the addition of suitable amounts of other nitrogen and phosphorous-containing yeast-nutrient materials substantially composed of ammonium sulphate and ammonium phosphte. This yeastnutrient solution then being seeded with a sufiicient amount of yeast of the species Saccharomyces cerevisiae, thoroughly aerated at a temperature of about 60 F. for the duration of the process, the temperature at least for the most part of the batch maintained during the process at a normal-propagative temperature of about 65 F.90 F. was revolved by a vaned impeller disposed near th bottom of an electrically-nonconductive vessel which is tall as compared to its width. As a cathode there was used the tubular stainless steel air distributor which is located inside and near the bottom of the vessel and to this tube is connected the negative lead of the current supply.

An acid constituent of the above described culture was removed by the employed of electro-endosmosis in the following manner: a hollow porous unglazed ceramic anode containing diaphragmatic section in the shape of a double-edged sword was installed in the lower half of the vessel being arranged horizontally and extending into the yeast-containing nutrient solution in a manner to be streamlined with said solution which was being revolved with a peripheral speed of about 300 feet per minute. This porous section containing the anode communicates with a vertical tubular anolyte reservoir arranged outside the vessel with a height above the level of the culture which also served as a catholyte. The anode consists of a carbon rod arranged to project down into the anolyte containing reservoir, thence horizontally into the finely porous anolyte containing ceramic section and to this anode is connected the positive lead of a direct current supply having a ripple and a potential of about -7 v. with about 4 /2 amp. per square foot of effective cathode surface. The porous diaphragrn surrounding the anode and anolyte is sufficiently dense to prevent the passage of yeast from the catholyte into the anolyte being confined in the anodic compartment or section of the cell thus preventing the contact of the culture with the anode and also providing a means of entrapping an acid constitutent of the yeast-nutrient solution which had migrated from the catholyte into the confined anolyte. The anolyte was composed of an aqueous solution of 5% ammonium sulphate and 5% potassium sulphate with a level in the anodic compartment slightly above the level of the culture.

As the propagation of yeast progressed, it being very desirable to maintain the acidity at the most optimum degree for the yeast, frequent acidity tests were made and when the acidity tended to unfavorably increase, this culture was treated with electro-endosmosis by the introduction of a current of electricity in the manner as above described until the undesirable degree of acid which included sulphuric acid was removed from the culture by the migration of anions into the anodic cell and therein confined. By the use of electro-endosmosis as described by this example the production of yeast rel ative to the weight of sugar used was twice that of an identical process wherein the removal of acidic constituents of the culture was not eifected by the employment of electro-endosmosis.

Example II For the employment of the present invention wherein lactic acid is produced by the action of a lactic acidproducing bacteria, a nutrient medium was prepared in the usual manner which consisted chiefly of hydrolized cane molasses with the addition of sufiicient nitrogen and phosphate containing salts. Ths nutrient solution was then inoculated with a suitable amount of the lactic acidproducing organisms L. delbrueckii and the temperature, at least for the most part of the batch, maintained during the process at a normal temperature of about 130 F. Also during the process the acidity was maintained at a maximum point which required from to cc. of a normal alkali solution to neutralize cc. of the inoculated-nutrient solution by removing the produced lactic acid with the employment of electro-endosmosis in the same manner and with the same equipment described in the above Example I, except that in this case an intermittent current of electricity was used and the anolyte was composed of an aqueous solution of 10% ammonium sulphate. After substantially all the sugar was converted the produced lactic acid was separated from the medium and anolyte by the well known method. By the use of electro-endosmosis as described by this example the production of lactic acid relative to the weight of the sugar used was more than twice that of an otherwise identical process wherein removal of lactic acid from the batch was not effected by electro-endosmosis.

Having now described my invention, I claim:

1. In a process for the growth of vegetative microorganisms that employs electrolysis with the use of an electrolytic solution, the improvement comprising the culture of said microorganisms when dialytically removing an ionized component from said solution by effecting electromotive migration of the ionized component and at the same time moving said culture with suflicient velocity to counteract electromotive migration of said microorganisms and so as to suspend them by said solution.

2. In a process for they growth of vegetative microorganisms that employs electrolysis with the use of an electrolytic solution, the improvement comprising the culture of said microorganisms when dialytically removing an ionized component in the form of anions having a decidedly acid character from said solution by efiecting their electromotive migration and at the same time moving said culture with sufficient velocity to counteract electromotive migration of said microorganisms and so as to suspend them by said solution.

3. In a process for the culture of vegetative microorganisms that employs electrolysis with the use of an electrolytic solution, the improvement comprising the culture of said microorganisms when dialytically removing an ionized component from said solution by effecting electromotive migration of the ionized component and at the same time moving said culture with sufiicient velocity to counteract electromotive migration of said microorganisms and so as to suspend them by said solution.

4. In a process for the culture of vegetative microorganisms that employs electrolysis with the use of an electrolytic solution, the improvement comprising the culture of said microorganisms when using a direct current of electricity selected from the group consisting of intermittent direct current and direct current which has a ripple and dialytically removing an ionized component from said solution by efiectirn electromotive migration of the ionized component and at the same time moving said culture with sufiicient velocity to counteract electromotive migration of said microorganisms and so as to suspend them by said solution.

5. In a process for the culture of vegetative microorganisms that employs electrolysis with the use of an electrolytic solution, the improvement comprising the culture of said microorganisms when dialytically removing an ionized component in the form of anions having a decidedly acid character from said solution by efi'ecting their electromotive migration and at the same time moving sa1d culture with sufiicient velocity to counteract electromotive migration of said microorganisms and so as to suspend them by said solution.

6. In a process for the growth of vegetative microorganisms in a fluid culture medium through which there is passed a current of electricity, the improvement which comprises the culture of said microorganisms when therefor passing a direct current of electricity through said culture selected from the group consisting of intermittent direct current and direct current having a ripple and at the same time moving said culture with suificient velocity to counteract electromotive migration of said microorganisms and so as to suspend them by said medium.

7. In a process for the culture of vegetative microorganisms in a fluid culture medium through which there is passed a current of electricity, the improvement which comprises the culture of said microorganisms when therefor passing a direct current of electricity through said culture selected from the group consisting of intermittent direct current and direct current having a ripple and at the same time moving said culture with suiiicient velocity to counteract electromotive migration of said microorganisms and so as to suspend them by said medium.

8. In a process for the growth of vegetative microorganisms that employs electrolysis with the use of an electrolytic solution, the improvement comprising the culture of said microorganisms when using a direct current of electricity selected from the group consisting of intermittent direct current and direct current which has a ripple and dialytically removing an ionized component from said solution by effecting electromotive migration of the ionized component and at the same time moving said culture with sufiicient velocity to counteract electromotive migration of said microorganisms and so as to suspend them by said solution.

References Cited in the file of this patent UNITED STATES PATENTS OTHER REFERENCES Biological Abstracts, vol. 3, article No. 17831, page 1668 (1929), by E. Benedetti.

Chemical Abstracts 28, 2839 (1934), article by Pirroni. Chemical Abstracts 28, 3830 (1934), article by Pirroni. 

1. IN A PROCESS FOR THE GROWTH OF VEGETATIVE MICROORGANISSMS THAT EMPLOYS ELECTROLYSISS WITH THE USE OF AN ELECTROLYTIC SOLUTION, THE IMPROVEMENT COMPRISING THE CULTURE IF SAID MICROORGANISMS WHEN DIALYTICALLY REMOVING AN IONIZED COMPONENT FROM SAID SOLUTION BY EFFECTING ELECTROMOTIVE MIGRATION OF THE IONIZED COMPONENT AND AT THE SAME TIME MOVING SAID CULTURE WITH SUFFICIENT VELOCITY TO COUNTREACT ELECTROMOTIVE MIGRATION OF SAID MICROORGANISMS AND SO AS TO SUSPEND THEM BY SAID SOLUTION. 